Methods of preventing ischemic injury using peripheral nociceptive stimulation

ABSTRACT

Methods of inhibiting ischemia-related and ischemia-reperfusion-related injury are provided. Remote administration of a C-fiber activator or TRPV1 agonist or remote electrical stimulation and activation of TRPV1 reduces ischemia-related tissue damage in subjects at risk for ischemia-related tissue damage. In aspects of the invention, remote application of a TRPV1 agonist inhibits ischemia-related cardiac tissue damage. Methods of inhibiting cardiac tissue damage by topically administering the TRPV1 agonist, capsaicin are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application No. 61/215,616, filed on May 7, 2009 and U.S.Provisional Patent Application No. 61/339,159 filed on Mar. 1, 2010,which are herein incorporated by reference in their entirety.

This application may be related to U.S. patent application Ser. No.12/800,110, entitled “METHODS OF PREVENTING ISCHEMIC INJURY USINGPERIPHERAL NOCICEPTIVE STIMULATION,” filed May 7, 2010, by Jones et al.,now U.S. Pat. No. 8,980,223, issued Mar. 17, 2015, the entire disclosureof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to the activation of TRP as a means ofinhibiting ischemia and reperfusion related tissue damage.

BACKGROUND OF THE INVENTION

Ischemia-related injury contributes significantly to morbidity andmortality throughout the world, with perhaps cardiac and cerebralischemia-related injuries being the most well-known (such as but notlimited to heart attack and stroke). Ischemic heart disease is a leadingcause of death in North America and is predicted to become moreprevalent as the population ages (Scroggins, 2001). Ischemia andreperfusion lead to tissue damage through a variety of mechanisms. Forexample, ischemia and reperfusion profoundly affect mitochondria and thecytosol. Current therapies for ischemic disease are directed at therestoration of blood flow to the ischemic region. However, duringreperfusion additional damage related to generation of reactive oxygenspecies occurs (Singh et al (1995) Mol Cell Biochem 147:77-81 andFlaherty et al (1998) Free Radic. Biol. Med. 5:409-419; hereinincorporated by reference in their entirety).

The transient receptor potential vanilloid-1 (TRPV1) is a capsaicinresponsive ligand-gated cation channel selectively expressed on small,unmyelinated peripheral nerve fibers (cutaneous nociceptors). SeeCaterina and Julius (2001) Annu Rev Neurosci 24:487-517 and Montell etal (2002) Mol. Cell. 9:229-231, herein incorporated by reference intheir entirety.

Capsaicin, a pungent substance derived from the plants of the solanaceaefamily (hot chili peppers) has been used as an experimental tool becauseof its selective action on the small diameter afferent nerve fibers,C-fibers and A-delta fibers that may be involved with signaling pain.Therapeutically capsaicin has been used as a topical analgesic. See forexample U.S. Pat. Nos. 4,997,853; 5,063,060; 5,178,879; 5,296,225;5,665,378; 6,248,788; 6,239,180; and 4,599,342; herein incorporated byreference in their entirety. Capsaicin binds and activates TRPV1.

SUMMARY OF THE INVENTION

Methods and kits for inhibiting ischemia- and/orischemia/reperfusion-related tissue damage in a subject are provided.The methods and kits are based on the development of means of providingtissue protection, particularly cardiac tissue protection by remotedelivery of a TRP agonist to subjects at risk for ischemia-relatedtissue damage. The methods and kits prevent or reduce ischemia andreperfusion related damage to tissues. Further the application providesmethods and kits for cardioprotection in subjects at risk forischemia-related cardiac tissue damage. Subjects at risk forischemia-related cardiac tissue damage include, but are not limited to,human subjects with one or more symptoms related to myocardial infarct,or heart attack. The methods and kits provide topical delivery of atherapeutically effective amount of a TRP agonist such as capsaicin to asubject at risk for ischemia-related tissue damage at predeterminedregions of the subject's body. Topical administration of the agonist ata predetermined region decreases ischemia-related tissue damage.

Methods and kits for inhibiting ischemia-related tissue damage in asubject are provided. The methods and kits are based on the developmentof means of providing tissue protection, particularly cardiac tissueprotection by remote activation of specific C-fiber nociception byelectrical stimulation such as but not limited to electro-acupuncture,to subjects at risk for ischemia-related tissue damage. The methods andkits prevent or reduce ischemia and reperfusion related damage totissues. Further the application provides methods and kits forcardioprotection in subjects at risk for ischemia-related cardiac tissuedamage. Subjects at risk for ischemia-related cardiac tissue damage mayinclude, but are not limited to, human subjects with one or moresymptoms related to myocardial infarct, or heart attack. Administrationof a therapeutically effective electrical stimulation at a predeterminedregion decreases ischemia-related tissue damage.

Methods of inhibiting ischemia-related tissue damage in a subject atrisk for ischemia-related tissue damage comprising the steps ofidentifying a subject at risk for ischemia-related tissue damage andtopically administering a therapeutically effective amount of a TRPV1agonist to a predetermined region of said subject are provided. In anembodiment the agonist is selected from the group comprising capsaicin,zucapsaicin, nonivamide, nicoboxil, davasaicin, piperine, rutaecarpine,capsaicinoids, resiniferotoxin, 3-hydroxyacetanilide, anandamide,α-acaridia, β-acaridial, pseudocapsaicin, dihydrocapsaicin,nordihydrocapsaicin anandamide, zingerone, warburganal, polygodial,aframodial, cinnamodial, cinnamosmolide, cinnamolide, isovelleral,scalaradial, ancistrodial, olvanil, merulidial, scutigeral, cannabidiol,civamide, N-arachidonoyldopamine, eugenol, guaiacol, vanillotoxins, andresiniferatoxin (RTX). In an embodiment, the agonist is capsaicin. In anaspect of the invention, the capsaicin consists essentially oftrans-capsaicin. In an aspect of the invention, the capsaicin consistsessentially of(6E)-N-[(4-Hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonenamide. Invarious aspects, the capsaicin is U.S.P.-grade. In various embodimentsthe therapeutically effective amount of capsaicin may be within therange of 10 μg to 200 mg capsaicin/kg of said subject. In an aspect ofthe methods, the therapeutically effective amount of capsaicin is withinthe range of 1 mg capsaicin/kg subject to 20 mg capsaicin/kg subject. Invarious embodiments the TRP agonist is administered in the form of anointment, cream, gel, patch, lotion, or spray.

In an embodiment, the subject is at risk for ischemia-related tissuedamage to one or more tissues. In aspects of the methods, the subject isat risk for ischemia-related damage to cardiac, cerebral, renal,intestinal, hepatic, splenic, ocular, retinal, pancreatic, pulmonary,vertebrobasilar or skeletal-muscle tissue. Such a subject is at risk forischemia-related cardiac, cerebral, renal, splenic, intestinal, hepatic,ocular, retinal, pancreatic, pulmonary; vertebrobasilar orskeletal-muscle tissue damage. In an aspect of the methods, the subjectis at risk for ischemia-related cardiac or cerebral tissue damage. In anaspect of the methods, the subject is at risk for ischemia-relatedcardiac tissue damage. In an aspect of the methods, the predeterminedregion for administering the agonist in a subject at risk forischemia-related cardiac tissue damage is selected from the group ofregions comprising the region extending from thirty centimeters superiorto the umbilicus and to thirty centimeters inferior to the umbilicus andencircling the subject and partial regions within the region extendingfrom thirty centimeters superior to the umbilicus and to thirtycentimeters inferior to the umbilicus and encircling the subject. In anaspect of the methods, the predetermined region contains sensory nervesfrom which signal travels to the dorsal root ganglia at or below the T7level. In an aspect of the methods, the subject is at risk forconditions that may include, but are not limited to, acute myocardialinfarction, angioplasty, cardiac arrest, cardiac surgery, cardiactransplantation or aneurism rupture.

In aspects of the methods the subject is at risk for ischemia-relatedcerebral tissue damage. Various aspects include methods where thesubject is at risk for ischemic stroke, transient ischemic stroke,hemorrhagic stroke, or aneurysm rupture.

Suitable subjects may include mammals, such as but not limited to,human, murine, equine, bovine, caprine, porcine, ovine, canine, orfeline mammals. In various aspects the agonist may be administered uponidentifying the subject as being at risk for ischemia-related cardiactissue damage, after identification of an ischemia-related symptom,concomitant with reestablishment of blood flow, or up to three hoursafter the reestablishment of blood flow. In an aspect, administering theagonist occurs within fifteen minutes of the first identification of anischemia-related symptom.

The current application provides methods of inhibiting ischemia-relatedcardiac tissue damage in a human subject comprising the steps ofidentifying a human subject at risk for ischemia-related cardiac tissuedamage and topically administering a therapeutically effective amount ofcapsaicin to a predetermined region selected from the group of regionscomprising the region extending from thirty centimeters superior to theumbilicus to thirty centimeters inferior to the umbilicus and encirclingthe subject, and one or more partial regions located with the regionextending from thirty centimeters superior to the umbilicus to thirtycentimeters inferior to the umbilicus and encircling the subject. In anaspect of the methods, administering the capsaicin occurs afteridentification of an ischemia-related symptom, concomitant withreestablishment of blood flow, or up to three hours afterreestablishment of blood flow. In an aspect, administering the capsaicinoccurs within fifteen minutes of the first identification of anischemia-related symptom. Various embodiments may include atherapeutically effective amount of capsaicin within the range of 10 μgto 200 mg capsaicin/kg subject.

An embodiment provides methods of modulating a cardiac tissue damagecharacteristic comprising the steps of identifying a subject at risk forischemia-related cardiac tissue damage; topically administering atherapeutically effective amount of a TRPV1 agonist to a predeterminedregion of the subject and evaluating a cardiac tissue damagecharacteristic. In an aspect of the embodiment, the TRPV1 agonist isselected from the group comprising capsaicin, zucapsaicin, nonivamide,nicoboxil, davasaicin, piperine, rutaecarpine, capsaicinoids,resiniferotoxin, 3-hydroxyacetanilide, anandamide, α-acaridial,β-acaridial, pseudocapsaicin, dihydrocapsaicin, nordihydrocapsaicinanandamide, zingerone, warburganal, polygodial, aframodial, cinnamodial,cinnamosmolide, cinnamolide, isovelleral, scalaradial, ancistrodial,olvanil, merulidial, scutigeral, cannabidiol, civamide,N-arachidonoyldopamine, eugenol, guaiacol, vanillotoxins, andresiniferatoxin (RTX). In an aspect of the embodiment, the TRPV1 agonistis capsaicin. In an aspect, the therapeutically effective amount ofcapsaicin is within the range of 10 μg to 200 mg capsaicin/kg of thesubject. In an aspect of the methods, administering the capsaicin occursafter identification of an ischemia-related symptom, concomitant withreestablishment of blood flow, or up to three hours afterreestablishment of blood flow. In aspects of the methods, the cardiactissue damage characteristic is a cardiac biomarker selected from thegroup comprising, but not limited to, troponin and creatine kinase. Inaspects of the methods the cardiac tissue damage characteristic isselected from the group comprising cardiac function and cardiacviability.

An embodiment provides methods of modulating the level of at least onecardiac biomarker in the serum of a subject at risk for ischemia-relatedcardiac tissue damage comprising the steps of identifying a subject atrisk for ischemia-related cardiac tissue damage, topically administeringa therapeutically effective amount of a TRPV1 agonist to a predeterminedregion of the subject, obtaining a serum sample from the subject andevaluating the level of at least one cardiac biomarker in the serumwherein the biomarker level is modulated as compared with an untreatedor placebo treatment effect. In an aspect of the method, the biomarkerlevel differs from the biomarker level in the serum of an untreated orplacebo-treated subject or population of subjects. In an aspect, thecardiac biomarker may include, but is not limited to, troponin orcreatine kinase.

An embodiment provides methods of modulating cardiac function in asubject at risk for ischemia-related cardiac tissue damage comprisingthe steps of identifying a subject at risk for ischemia-related cardiactissue damage, topically administering a therapeutically effectiveamount of a TRPV1 agonist to a predetermined region of the subject andperforming at least one imaging modality on the subject, wherein theimaging modality indicates the cardiac function is modulated as comparedwith a similar subject receiving placebo therapy. In aspects of themethods, the imaging modality may include, but is not limited to,echocardiography, nuclear imaging, or magnetic resonance imaging. Inaspects of the methods, the cardiac function is improved as comparedwith a subject receiving placebo therapy.

The current application provides kits for use in preventingischemia-related damage comprising a TRP family receptor agonist suchas, but not limited to, a TRPV1 agonist, in a unit dose form for topicaladministration and usage instructions. In aspects of the kit, the TRPV1agonist is selected from the group comprising capsaicin, zucapsaicin,nonivamide, nicoboxil, davasaicin, piperine, rutaecarpine,capsaicinoids, resiniferotoxin, 3-hydroxyacetanilide, anandamide,α-acaridial, β-acaridial, pseudocapsaicin, dihydrocapsaicin,nordihydrocapsaicin anandamide, zingerone, warburganal, polygodial,aframodial, cinnamodial, cinnamosmolide, cinnamolide, isovelleral,scalaradial, ancistrodial, olvanil, merulidial, scutigeral, cannabidiol,civamide, N-arachidonoyldopamine, eugenol, guaiacol, vanillotoxins, andresiniferatoxin (RTX). In an aspect, the agonist is capsaicin. In anaspect of the kit, the kit provides the agonist in the form of anointment, cream, gel, patch, lotion, liquid, or spray. In an aspect ofthe kit, the usage instructions provide a predetermined region foradministration of the agonist to the subject. In an embodiment of thekit, the kit is for use in preventing ischemia-related cardiac tissuedamage. In an aspect of the embodiment wherein the kit is for use inpreventing cardiac tissue damage, the predetermined region is a regionextending from thirty centimeters superior to the umbilicus to thirtycentimeters inferior to the umbilicus and encircling the subject or apartial region within that region.

Methods are provided for inhibiting ischemia-related injury during organtransplantation. The methods comprise topically administering to atleast one of the donor and recipient subjects a therapeuticallyeffective amount of a TRPV1 agonist at a predetermined location prior toor concomitant with the organ transplantation procedure.

The application provides methods of inhibiting ischemia-related injuryin a mammalian donor organ, comprising topically administering to thedonor subject a therapeutically effective amount of a TRPV1 agonist at apredetermined location prior to the organ transplantation procedure.

The application provides methods of inhibiting ischemia-related tissuedamage in a subject at risk for ischemia-related damage comprising thesteps of: identifying a subject at risk for ischemia-related tissuedamage; and topically administering a therapeutically effective amountof a C fiber stimulator. In aspects of the methods, the C fiberstimulator is a TRP family (including but not limited to the TRPV, TRPA,TRPM, TRPP, TRPC subfamilies, each of which has multiple members)receptor agonist, a TRPV1 agonist, capsaicin, capsaicin analogue, orelectrical stimulation.

Methods are provided for inhibiting ischemia-related tissue damage in asubject at risk for ischemia-related tissue damage comprising the stepsof identifying a subject at risk for ischemia-related tissue damage andtopically administering a therapeutically effective electricalstimulation to a predetermined region of the subject. In aspects, theelectrical stimulation may include electro-acupuncture. In variousaspects, the subject is at risk for ischemia-related tissue damage thatmay include cardiac, cerebral, renal, pulmonary, intestinal, hepatic,pancreatic, splenic, ocular, retinal, vertebrobasilar, orskeletal-muscular tissue damage. In an aspect, the subject is at riskfor ischemia-related cardiac tissue damage. In embodiments wherein thesubject is at risk for ischemia-related cardiac tissue damage, thepredetermined region may be selected from the group of regionscomprising the region extending from thirty centimeters superior to theumbilicus to thirty centimeters inferior to the umbilicus and encirclingthe subject and at least one partial region located with the regionextending from thirty centimeters superior to the umbilicus to thirtycentimeters inferior to the umbilicus and encircling the subject. In anaspect of the methods, the predetermined region contains sensory nervesfrom which signal travels to the dorsal root ganglia at or below the T7level.

In various embodiments the subject may be at risk for acute myocardialinfarction, angioplasty, cardiac arrest, cardiac surgery, cardiactransplantation, or aneurism rupture. In various aspects of the methods,administering the electrical stimulation occurs upon identifying thesubject as being at risk for ischemia-related cardiac tissue damage,after identification of an ischemia-related symptom, concomitant withreestablishment of blood flow or up to three hours after reestablishmentof blood flow. In aspects of the methods, administering the electricalstimulation occurs within fifteen minutes of the first identification ofan ischemia-related symptom.

In an embodiment, methods of modulating an angina related symptom in asubject at risk for angina are provided. The methods involved the stepsof identifying a subject at risk for angina and topically administeringa therapeutically effective amount of a TRP family receptor agonist to apredetermined region of the subject. In an aspect of the methods, theagonist is administered chronically.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 presents a series of panels describing the murineischemia/reperfusion model of myocardial infarction and the effects ofremote (abdominal) topical nociceptive stimulation on myocardialinfarction. These data demonstrate that remote application of either theTRPV1 agonist capsaicin or electrical stimulation of TRPV1 inhibitsischemia-related cardiac tissue damage. Panel A presents images of amurine heart prior to induction of ischemia (A1, pre-ischemia), aftersurgical induction of ischemia by coronary occlusion (A2, ischemia), andafter reestablishment of blood flow (A3, reperfusion) andechocardiographs patterns. Cyanosis was present during ischemia (middleframe, A2). After reperfusion (right frame, A3), cardiac tissuecoloration is visibly altered (blushing) compared to cyanotic, ischemictissue. The hearts were continuously monitored by electrocardiograph(ECG) during the procedure and tracings during each phase are presentedbeneath the photograph panels (FIG. 1A4 and FIG. 1A5). Voltage (y-axis)is measured in milliVolts; time x-axis) is measured in seconds. Duringthe ischemic portion of the experiment, the ST segment is elevated (A4).Upon reperfusion, the ECG tracing changed from that seen during ischemia(A5). An ECG tracing at 5 minutes after reestablishment of blood flow(reperfusion +5 minutes, A5) is also presented. By 5 minutes postreperfusion, the ECG tracing had changed further. The ECG tracingssupport this in vivo mouse model of ischemia and reperfusion (I/R).

FIG. 1, panel B presents results obtained from mice were subjected toeither remote abdominal electrical stimulation or sham stimulation priorto coronary occlusion. Panels 1B(1) and 1B(2) are triphenyltetrazoliumchloride (TTC) stained images of sections of murine hearts taken after45 minutes of coronary occlusion (CO) and reperfusion showing myocardialinfarction. Panel 1B(1) (Nociceptive Stimulus) shows a myocardialinfarction in a mouse that received nociceptive stimulation with eitherabdominal transcutaneous electrical stimulation or chemical stimulationwith capsaicin prior to coronary occlusion and induction of ischemia.Panel 1B(2) shows a myocardial infarct from a sham treated mouse (Shamcontrol). Red tissue is living, white tissue is dead, and blue tissuewas not in the ischemic region.

FIG. 1, panel C presents a graph summarizing data obtained from mice(n=6) that underwent either electrical stimulation (ES), administrationof capsaicin (Capzasin), or sham treatments (Sham) prior toischemia/reperfusion. Infarct size as a percent of the risk region ispresented on the y-axis. Data from sham-treated mice are presented withhatched bars; data from electrically stimulated or capsaicin treatedmice are presented with empty bars (nociceptive stimulus). Remotepre-treatment with either capsaicin or electrical stimulation resultedin a substantial reduction in infarct size. In this experiment, remotepre-conditioning (stimulus given 15 minutes prior to the ischemia)reduced the infarct size by 85%. Asterisks indicate P<0.01 compared tothe sham values. Values are mean±s.e.m.

FIG. 1, panel D presents a graph summarizing results obtained fromsham-treated mice and mice stimulated after induction of ischemia(post-CON). Infarct size as a percent of the risk region is presented onthe y-axis. Data from sham-treated mice are presented with hatched bars;data from electrically stimulated mice are presented with cross-hatchedbars. Infarct size as a percentage of risk region was significantlyreduced in mice that received electrical stimulation during the ischemicperiod. Further details are provided in example 13. Asterisks indicateP<0.01 compared to the sham values. Values are mean±s.e.m.

FIG. 1, panel E presents photomicrographs of cardiac tissue sectionsobtained from untreated (normal myocardium, 1E(1)), pre-treated(nociceptive stimulus, 1E(2)) and sham-treated mice (sham control,1E(3)). The tissue sections were stained with hematoxylin and eosin (H&Estaining) prior to imaging. Note the dropout of myocardial cells andinfiltration of mononuclear cells, both indicative of ischemia relatedtissue injury in the sham panel.

FIG. 1, panel F presents photomicrographs of cardiac tissue sectionsobtained from untreated (normal myocardium, (1F(1)), pre-treated(nociceptive stimulus (1F(2)) and sham-treated mice (sham control(1F(3)). The tissue sections were TUNEL stained prior to imaging. TUNELpositive nuclei fluoresce brightly in this assay and are indicative ofprogrammed cell death (apoptosis). Tissue from sham-treated mice showssignificant TUNEL-positive nuclei (bright white regions) while fewer arepresent in tissue from pre-treated mice. The elevated TUNEL staining inthe sham treated cells indicated elevated apoptosis while the reducedlevel of TUNEL staining in the pre-treated cells indicated a reducedlevel of apoptosis compared to the sham-treated cells.

FIG. 1, panel G presents a graph summarizing the percentage of apoptoticnuclei in tissue samples from pre-treated and sham-treated mice. FIG. 1,panel H presents a graph summarizing the percentage of DNA fragmentation(also indicative of apoptosis) in tissue samples from pre-treated andsham-treated mice. Data from sham-treated mice are presented withhatched bars; data from electrically stimulated mice are presented withcross-hatched bars (nociceptive). The number of TUNEL positive nucleiand the DNA fragmentation were significantly reduced in the pre-treatedgroup, compared to the sham-treated group (P<0.001, n=4). These dataprovide evidence that pre-treatment reduces apoptosis and inhibitsischemia-related tissue damage.

FIG. 2 presents panels summarizing data obtained from experimentsevaluating C-fiber neural involvement and consequent molecular signalingin the inhibition of ischemia-related tissue damage. Panel A presents agraph of infarct size as a percent of risk region (y-axis) ofsham-treated (no ES, control) or electrically stimulated (ES) mice. Thedata in the first column (hatched bar) are from control mice thatreceived neither advance treatment nor electrical stimulation prior toinduction of ischemia. The data in the middle column (cross-hatched bar)are from mice treated with vehicle prior to electrical stimulation andinduction of ischemia (vehicle ES). The data in the last column (dottedbar) are from mice treated with 2% lidocaine prior to electricalstimulation and induction of ischemia (lidocaine ES). Infarction size inlidocaine and ES treated mice shows a significantly higher percentage ofrisk region than infarction size in vehicle and ES treated mice. Topicallidocaine inhibits nociceptive stimulation of C-fibers and appears toinhibit the cardioprotective effect; thus, nociception of C-fibers at apredetermined location may be involved in eliciting cardioprotection andinhibition of ischemia-related tissue damage.

Panel B presents a graph of infarct size as a percent of risk region inmice subjected to either spinal cord transection (transection) or shamspinal transection where the spinal cord remained intact (sham). Datafrom mice where the transection or sham transection was at cervicalvertebra 7 (C7) are indicated with empty bars. Data from mice where thetransection or sham transection was at thoracic vertebra 7 (T7) areindicated with hatched bars. Immediately after the spinal procedure,mice were subjected to abdominal incision, induction of ischemia andreperfusion. Infarct size was determined 4 hours later. Infarct size asa percentage of the risk region remained significantly high in mice withspinal transection at T7. Asterisks indicate P<0.01 compared to the shamvalues. Values are mean±s.e.m. Surgical transection at T7 preventscardioprotection, suggesting that intact spinal nerves to T7 may berequired for the stimulus to trigger protection of cardiac tissue.Transection at C7 does not significantly impact cardioprotectionsuggesting that the central nervous system may have a limited role incardioprotection through TRP family receptor activation.

FIG. 2, panel C depicts confocal micrographs of sectioned spinal cord(40 μm) (upper row, 2C(1)-(3)) and whole mount dorsal root ganglia(lower row, (2C(4)-(6)). The formaldehyde fixed spinal cord and dorsalroot ganglia at thoracic vertebra T9-T10 (FIG. 2C(3) and FIG. 2C(6) andT1-T5 (FIG. 2C(2) and FIG. 2C(5)) levels were dissected. One week priorto fixation, the mice were injected subcutaneously with a fluorescentdye 1,1′-dioactadecyl-3,3,3′,3′-tetramethylindocarbodyanine perchlorate(Dil) at the abdominal incision level (near the level of thoracicvertebra T9-T10 of the spine). After one week, dye labeled sensoryneurons and spinal motoneurons at the T9-10 level and neurons at thevertebral T1-T5 level. While not being limited by a mechanism, thesedata may suggest active neural pathways from the skin sensory nerves tothe spine, and from one level of the spine dorsal horn to higher levels,particularly to the T1-5 level, where the cardiac nerves connect to thespinal nerves. This may be a neural pathway from the sensory nerveendings (C-fibers) to the cardiac nervous system. While not beinglimited by mechanism, the stimulus may activate spinal nerves in thedorsal horn at the level of the incision, these neurons may activatethose in the dorsal horn of spinal segments superior, and the signal maytravel antidromically along the dorsal root and may activate the sensoryfibers innervating the heart (dorsal root reflex).

FIG. 2, panel D presents a graph of results obtained from wild-type (WT)and TRPV1 knock-out (TRPVKO) mice. Infarct size as a percent of riskregion is presented on the y-axis. The first bar presents data fromwild-type mice subjected to electrical stimulation (WT ES, hatched)prior to surgical induction of ischemia and reperfusion. The second barpresents data from TRPV1 knockout mice subjected to electricalstimulation (TRPVKO ES, dotted) prior to surgical induction of ischemiaand reperfusion. The third bar presents data from TRPV1 knockout micesubjected to sham stimulation (TRPVKO no-ES, empty) prior to surgicalinduction of ischemia and reperfusion. The infarct size as a percent ofrisk region in TRPV1 knockout mice subjected to electrical stimulationis significantly greater than the infarct size of wild-type micesubjected to electrical stimulation. The reduction of thecardioprotective effect in TRPV1 knockout mice may indicate involvementof TRPV1, a capsaicin sensitive C-fiber cation channel, in inhibition ofischemia-related tissue damage.

FIG. 2, panel E presents a graph of results obtained from wild-type (WT)and TRPV1 knock-out (TRPVKO) mice using topical capsaicin as thecardioprotective stimulus. Infarct size as a percent of risk region ispresented on the y-axis. The first bar presents data from wild-type micetopically treated with capsaicin administered to the abdomen prior tosurgical induction of ischemia and reperfusion (WT, Capsaicin, hatchedbar). The second bar presents data from TRPV1 knockout mice topicallytreated with capsaicin administered to the abdomen prior to surgicalinduction of ischemia and reperfusion (TRPVKO, Capsaicin, dotted bar).The third bar presents data from TRPV1 knockout mice subjected to shamtreatment prior to surgical induction of ischemia and reperfusion(TRPVKO No capsaicin, empty bar). Wild-type mice treated with capsaicinhave reduced myocardial infarct size as compared to TRPVKO micepotentially indicating TRPV1 involvement in inhibition ofischemia-related tissue damage by capsaicin.

The panels of FIG. 3 present data from experiments assessing involvementof additional receptors and mediators in the cardioprotective effectbrought about by administration of electrical stimulation or a TRPV1agonist such as capsaicin. Panel A presents data from wild-type (WT) andbradykinin receptor 2 knockout (BK2RKO) mice subjected to theischemia/reperfusion protocol described elsewhere herein. Mice wereeither sham-treated (control, hatched or cross hatched bars) or wereelectrically stimulated (ES, dotted or empty bars) as describedelsewhere herein. Infarct size as a percentage of risk region ispresented on the y-axis. Electrical stimulation of wild-type micesignificantly reduces the infarct size relative to control wild-type.Electrical stimulation of the bradykinin receptor 2 knockout miceinduces no significant reduction of infarct size relative tosham-treated BK2RKO mice. While not limited by mechanism, BK2R may beinvolved in the inhibition of ischemia-related tissue damage. BK2R isknown to be expressed in sympathetic nerve cells and cardiomyocytes.

Panel B presents data from wild-type mice treated with propanolol orvehicle alone. The mice then underwent either sham treatment orelectrical stimulation (ES) followed by the ischemia/reperfusionprocedure. Propanolol is relatively specific β-adrenergic receptorantagonist. The hatched bar presents data from mice treated withpropanolol and subject to sham treatment (propanolol control). Thedotted bar presents data from mice treated with vehicle alone andelectrically stimulated (vehicle ES). The cross-hatched bar presentsdata from mice treated with propanolol and electrically stimulated(propanolol ES). Infarct size as a percentage of risk region ispresented on the y-axis. Infarct size in mice treated with propanololand electrically stimulated is significantly higher than infarct size incontrol mice treated only with the vehicle and electrically stimulated.Blockade of the β-adrenergic receptor reduces development ofcardioprotection suggesting involvement of cardiac sympathetic nerves ininhibition of ischemia-related tissue damage.

Panel C presents data from wild-type mice treated with chelerythrineprior to sham treatment or electrical stimulation followed by theischemia/reperfusion procedure. Chelerythrine is a relatively selectivegeneral protein kinase C (PKC) inhibitor. Most previously describedforms of preconditioning or postconditioning require PKC activation incardiomyocytes. Typically this is indicative of activation and/orrepression of specific PKC isoforms in the hearts. Infarct size as apercentage of risk region was assessed and is indicated. The hatched barpresents data from mice treated with chelerythrine and subject to shamtreatment (chelerythrine control). The dotted bar presents data frommice treated with vehicle alone and electrically stimulated (vehicleES). The cross-hatched bar presents data from mice treated withchelerythrine and electrically stimulated (chelerythrine ES). In thisexperiment, inhibition of PKC resulted in reduced inhibition ofischemia-related tissue damage in the chelerythrine and electricallystimulated mice as compared to the vehicle alone and electricallystimulated mice. While not being limited by mechanism, PKC may beinvolved in the inhibition of ischemia-related tissue damage.

Panel D presents images of Western blotting results on the cytosolic(Cy) and membrane (M) fractions of myocardium. Mice underwent eithersham treatment or electrical stimulation. The membranes were incubatedwith antibodies specific to PKCα, PKCε or PKCδ.

FIG. 4 presents a graph of infarct size as a percentage of risk regionin mice subjected to the surgical ischemia and reperfusion protocoldescribed elsewhere herein. Infarct size is on the y-axis. The hatchedbar summarizes data from mice that received sham treatment only (NoRPCT). The dotted bar summarizes data from mice subjected to anabdominal wall incision prior to ischemia and reperfusion (WallIncision). The cross-hatched bar summarizes data from mice subjected toan abdominal skin incision prior to ischemia and reperfusion (SkinIncision). Mice pretreated with either an abdominal wall incision or askin incision exhibited reduced cardiac tissue damage as reflected bythe reduced infarct size.

FIG. 5 depicts a cartoon schematic of a possible model of the dorsalroot reflex that may be related to the current methods of inhibitingischemia-related cardiac tissue damage by remotely activating a TRPfamily receptor. DRG refers to the dorsal root ganglia. BK refers tobradykinin. NE refers to norepinephrine. SP refers to substance P. CRGPrefers to calcitonin gene related peptide (CGRP). A nociceptive stimulusby a TRP family receptor agonist, TRPV1 receptor agonist, a TRPV1agonist, capsaicin, capsaicin analogue, electrical stimulation, incisionor other mechanism, may activate a C-fiber sensory nerve, the signal maytravel to the dorsal root ganglia at that level and then up the spineperhaps via the dorsal root reflex. At the level of cardiac innervation,the signal may move to the heart, the cardiac sensory nerves may releaseSP and CGRP and may activate the cardiac sympathetic nerves. The cardiacsympathetic nerves may then release BK and NE which may then bindreceptors on the cardiomyocytes.

FIG. 6 depicts a possible model of the further aspects of the role ofremote C-fiber stimulation in cardioprotection againstischemia/reperfusion related injury. C-fiber stimulation may trigger anerve impulse in sensory nerves in the heart that may lead to release ofneurotransmitters such as CGRP (calcitonin gene related peptide) andsubstance P. These neurotransmitters may bind receptors activatingsympathetic nerves which may then release norepinephrine (NE) andbradykinin (BK). NE may bind to its receptor β-adrenergic receptor(β-AR) and BK may bind to its receptor BK2R, both of which are presenton cardiomyocytes. Binding of either or both of these receptorsactivates or represses specific PKC isoforms and may activate theK_(ATP) channels. The sum result of the modulation of these signalingpathways and effectors may be cardioprotection againstischemia/reperfusion injury.

FIG. 7, panels A-D present quantitative analysis of Western blots usingantibodies specific to PKCα (Panel A), PKCε (Panel B), PKCδ (Panel C),and PKCζ (Panel D). The ratio of the cytosolic fraction of the proteinto the membrane fraction of the protein is indicated on the y-axis ineach graph (Membrane cyoplasmic ratio). Dotted bars indicate resultsfrom sham treatment (sham); hatched bars indicate results fromelectrical treatment (APC). There is an increase in translocation ofPKCα and PKCε and a decrease in PKCδ in electrically stimulated cells.This is consistent with the prevailing evidence that PKCε and PKCα maybe protective while PKCδ may be injurious.

FIG. 8 presents a schematic of pre and postconditioning time lines byway of example.

FIG. 9 presents a schematic representation of the course of human STelevation MI (STEMI) along the bottom and topical administration of aTRPV receptor family agonist in mice in the upper two bars. Thetimeframe of the topical administration is indicated with a hatched bar.The post-reperfusion time frame is indicated with back-hatched bars(Reperfusion). The diagram indicates the relevance of the miceexperiments to human clinical STEMI as compared to postconditioning.

FIG. 10 presents a graph of infarct size as a percent of risk area inmice that received topical capsaicin application (duringischemia=conditioning; after reperfusion=postCON) (empty bars) or shamtreatments (hatched bars). Asterisks indicate significant results(p<0.01). Topical capsaicin significantly reduces infarct size.

FIG. 11 presents a graph of infarct size as a percent of risk area inmice that received topical probenecid application (empty bars) or shamtreatments (hatched bars). Infarct size as a percent of risk region issignificantly reduced in mice treated with probenecid, a TRPV2 agonist,compared to control animals (p<0.07).

FIG. 12 presents a graph of infarct size as a percent of risk area inmice that received either a sham treatment (sham, hatched bar) or anelectrical stimulation (late NIC, empty bar) twenty four hours prior tocoronary occlusion/reperfusion. The infarct size as a percent of riskregion in mice that received the electrical stimulation wassignificantly reduced compared to sham treated mice (p≦0.001). Theelectrical stimulation was applied to the skin in a region below thearea corresponding to the T7 vertebral level.

The schematic at the top of the figure represents the timeline of a typeof positive control for cardioprotection using multiple, small,non-lethal periods of ischemia (6×CO-R) prior to a 30 minute ischemicperiod (CO-R) prior to determination of infarct size (infarct size).

DETAILED DESCRIPTION OF THE INVENTION

Remote stimulation of a C fiber provides a protective effect at distanttissues or organs at risk for ischemia-related tissue damage. C fiberstimulation may result from administration of a TRP family receptoragonist, TRPV family receptor agonist, a TRPV1 agonist or electricalstimulation. More particularly, topical administration of a TRPV1agonist to a predetermined region of a subject inhibits ischemia-relatedtissue damage. Yet more particularly, topical administration ofcapsaicin inhibits ischemia-related cardiac tissue damage.

“Inhibiting” means partially or completely blocking, reducing,preventing, lessening, diminishing, or decreasing a particular processor activity. Inhibiting a particular process or activity may be a 0.01%,0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%,16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%,44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%,72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, orup to a 100% decrease in the process or activity. Evaluating inhibitingmay involve evaluating a result or effect of a particular process oractivity.

Ischemia-related tissue damage may include, but is not limited to, celldeath, apoptosis, increased inflammation, increased infiltration,mitochondrial damage, acidosis, increased inorganic phosphate, elevatedcalcium, altered tissue function, altered tissue viability, alteredcardiac function, altered cardiac viability, altered cerebral function,altered cerebral viability, and increased long-chain acyl coenzyme A.Methods of evaluating ischemia-related tissue damage may include, butare not limited to, computed tomography (CT), noncontrast CT, magneticresonance imaging (MRI) scanning, arteriography, and lumbar puncture.

Tissue types that may experience ischemia-related damage may include,but are not limited to, cardiac, cerebral, ocular, retinal, renal,pulmonary, intestinal, hepatic, pancreatic, splenic, vertebrobasilar,and skeletal-muscular tissues. Tissue types of particular interest mayinclude, but are not limited to, cardiac and cerebral tissue.

Suitable subjects may include mammals, such as but not limited to,human, simian, murine, equine, bovine, caprine, porcine, ovine, canine,or feline mammals, mammalian domestic livestock, mammalian companionanimals, and mammalian zoo, circus, and research animals.

A subject “at risk for” a particular condition may include subjectsexhibiting at least one symptom associated with the condition, subjectsexhibiting at least on clinical symptom associated with the condition,subjects in the process of experiencing the condition, and subjectsexperiencing the condition. A subject at risk for ischemia-relatedtissue damage may exhibit at least one symptom associated with acondition that results in ischemia, may exhibit at least one clinicalsymptom associated with a condition that results in ischemia, may be inthe process of experiencing a condition that results in ischemia, mayexhibit at least one symptom associated with ischemia, may exhibit atleast one clinical symptom associated with ischemia, or may beexperiencing ischemia. Ischemia-related tissue damage may include tissuedamage that results directly from the ischemic incident, tissue damagethat results from reperfusion after an ischemic period, and tissuedamage that occurs during reperfusion after an ischemic period. Ischemiais a low oxygen state that may result from causes such as but notlimited to, obstruction of arterial blood supply or inadequate bloodflow leading to hypoxia in the tissue.

Ischemia-related symptoms or symptoms associated with ischemia mayinclude, but are not limited to, sudden severe headache; sudden numbnessor weakness of the face, arm or leg, especially on one side of the body;sudden confusion, aphasia, loss of function of extremities, paralysis ofextremities; sudden trouble seeing in one or both eyes; suddendizziness, trouble walking, loss of balance, loss of coordination;facial flaccidness, loss of facial expression, unequal pupil size,tachycardia, coma, incontinence, nausea, convulsions, hypotonia,dysarthria, unresponsive, absence of pulse, cyanosis, dyspnea, coughwith sputum, syncope, anxiety, depression, feeling of impending doom,tachycardia, brachycardia; arrhythmia; asystole; ventricularfibrillation, chest pain; pain in arms, neck or jaw; diaphoresis, lossof consciousness, reduced ejection fraction, rales, cold, gallop rhythm,oliguria, anuria, abnormal chest x-ray, dysrrhythmia, altered cardiacenzymes, altered cardiac biomarkers, mixed venous O₂, reduced capillaryrefill, use of accessory muscles, nasal flaring, palpitations, edema,altered vision, light flashes in the visual field, vitreous floaters,loss of sight, blurred vision, loss of visual acuity, hypertension,hypotension, hematuria, elevated BUN/creatinine ratio, and urge to void.

Angina related symptoms may include, but are not limited to, chestdiscomfort, chest pain, dyspnea, stress-induced ST depression,reversible perfusion abnormality, or wall motion abnormality.

Methods of analyzing, evaluating, assessing or measuring an anginarelated symptom are known in the art. Such methods may include, but arenot limited to exercise tolerance testing (ETT), stress myocardialperfusion imaging, stress echocardiography, ETT with and withoutTechnetium Tc99m Tetrofosmin single photon emission computed tomography(SPECT). Functional classification of angina methods include, but arenot limited to, the Canadian Cardiovascular Society FunctionalClassification of Angina and modifications to include exercisetolerance. See for example Hackett & Cassem (1978) Rehabilitation of theCoronary Patient, John Wiley & Sons p. 243-253 and Shubb et al (1996)Mayo Clinic Practice of Cardiology 3^(rd) Ed. Mosby, p. 1160-1190;herein incorporated by reference in their entirety.

Identifying, noticing, observing, or recognizing a subject that is atrisk for a condition may include observing that the subject isexperiencing one or more symptoms related to the condition or one ormore symptoms related to a disease or disorder related to the condition.Thus, identifying a subject at risk for ischemia-related tissue damagemay involve identifying an ischemia-related symptom or identifying asymptom of an ischemia-related condition in the subject. It isenvisioned that the methods and kits encompass identification of asymptom of an ischemia-related condition such as heart attack or strokeby the care-provider (such as but not limited to, a first-responder,emergency medical technician, paramedic, layman, clinician) or subjecteven in instances where the care provider or subject does not fullyunderstand the physiological pathway.

The methods and kits of the invention are useful for the treatment of avariety of diseases, disorders, and conditions that involveischemia-related tissue damage. Ischemia-related tissue damage may bethe basis of a disease, disorder, or condition; may result from adisease, disorder, or condition; or may result from an injury, trauma,tear, or surgical procedure. As used herein, “disease”, “disorder”, and“condition” may be used interchangeably. Conditions wherein there is arisk of ischemia-related tissue damage to a subject or ischemia-relatedconditions may include, but are not limited to, reperfusion orrevascularization therapy, aneurism rupture, injury and trauma resultingin a significant decrease in blood volume such as but not limited tosevering of limbs or digits and replantation thereof, acute myocardialinfarction, cardiac infarction, heart attack, cardiac arrest, electiveangioplasty, angioplasty, coronary artery bypass graft, cardiac surgery,cardiac bypass, organ transplantation such as cardiac transplantation,head trauma, sepsis, cardiac arrest, drowning, shock, hemorrhage,anaphylaxis, a crush injury, angina, heart failure, cardiovascularcollapse, pneumothorax, embolism, decompression sickness, sudden cardiacarrest, SCA, congestive heart failure, myocardial ischemia, coronaryartery disease, stroke, cerebral infarction, brain attack, CVA,cerebrovascular accident, transient ischemic attack, transient ischemicstroke, intracranial hemorrhage, cerebral emboli, acute ischemic stroke,hemorrhagic stroke, mini-stroke, ischemic stroke, renal ischemia,ischemic ocular neuropathy, retinal break, retinal detachment, warmischemia of transplant organs or tissues, stress induced angina, anginapectoris, chronic angina, stable angina, and chest pain.

A subject at risk for ischemia-related cardiac tissue damage may be atrisk for a condition such as but not limited to, reperfusion orrevascularization therapy, aneurism rupture, injury and trauma resultingin a significant decrease in blood volume such as but not limited tosevering of limbs or digits and replantation thereof, acute myocardialinfarction, cardiac infarction, heart attack, cardiac arrest, electiveangioplasty, angioplasty, coronary artery bypass graft, cardiac surgery,cardiac bypass, organ transplantation such as cardiac transplantation,head trauma, sepsis, cardiac arrest, drowning, shock, hemorrhage,anaphylaxis, a crush injury, angina, heart failure, cardiovascularcollapse, pneumothorax, embolism, decompression sickness, sudden cardiacarrest, SCA, congestive heart failure, myocardial ischemia, stressinduced angina, angina pectoris, chronic angina, stable angina, chestpain and coronary artery disease.

A subject at risk for ischemia-related cerebral tissue damage may be atrisk for a condition such as but not limited to aneurism rupture,stroke, cerebral infarction, brain attack, CVA, cerebrovascularaccident, transient ischemic attack, transient ischemic stroke,intracranial hemorrhage, cerebral emboli, acute ischemic stroke,hemorrhagic stroke, mini-stroke, and ischemic stroke.

Methods of modulating angina related symptoms may relieve, ameliorate,improve, alter, change, reduce, or alleviate an angina related symptom.It is recognized that the methods of the instant application may beutilized in conjunction with other methods of modulating an anginarelated symptom known in the art that may include administeringnitrates, beta-blockers, calcium channel antagonists, ranolazine,aspirin, platelet inhibitors, risk factor modification, or surgery.

Methods and kits of the current application provide for topicaladministration of an agent to a pre-determined region of a subject atrisk for ischemia-related tissue damage. A pre-determined region may bean anatomical region of the subject such as a defined, designated,limited, particular or specific area of skin, location of skin, portionof skin, or site. It is recognized that a predetermined region suitablefor use with a subject at risk for ischemia-related damage of anindicated tissue may or may not be suitable for use with a subject atrisk for ischemia-related damage of a different indicated tissue.Pre-determined regions suitable for use on subjects at risk forischemia-related cardiac tissue may include, but are not limited to, theupper chest, the arm, the abdomen, the lower abdomen, the upper abdomen,the region extending from thirty centimeters superior to the umbilicusto thirty centimeters inferior to the umbilicus and encircling thesubject, and partial regions with in the region extending from thirtycentimeters superior to the umbilicus to thirty centimeters inferior tothe umbilicus and encircling the subject. Additional suitable regionsfor use with subjects at risk for ischemia-related cardiac tissue damagemay located within a region extending from 60 cm superior to theumbilicus to 50 cm inferior to the umbilicus, 50 cm superior to theumbilicus to 50 cm inferior to the umbilicus, 40 cm superior to theumbilicus to 40 cm inferior to the umbilicus, 45 cm superior to theumbilicus to 45 cm inferior to the umbilicus, 40 cm superior to theumbilicus to 40 cm inferior to the umbilicus, 35 cm superior to theumbilicus to 35 cm inferior to the umbilicus, 30 cm superior to theumbilicus to 30 cm inferior to the umbilicus, 29 cm superior to theumbilicus to 29 cm inferior to the umbilicus, 28 cm superior to theumbilicus to 28 cm inferior to the umbilicus, 27 cm superior to theumbilicus to 27 cm inferior to the umbilicus, 26 cm superior to theumbilicus to 26 cm inferior to the umbilicus, 25 cm superior to theumbilicus to 25 cm inferior to the umbilicus, 24 cm superior to theumbilicus to 24 cm inferior to the umbilicus, 23 cm superior to theumbilicus to 23 cm inferior to the umbilicus, 22 cm superior to theumbilicus to 22 cm inferior to the umbilicus, 21 cm superior to theumbilicus to 21 cm inferior to the umbilicus, 20 cm superior to theumbilicus to 20 cm inferior to the umbilicus, 19 cm superior to theumbilicus to 19 cm inferior to the umbilicus, 18 cm superior to theumbilicus to 18 cm inferior to the umbilicus, 17 cm superior to theumbilicus to 17 cm inferior to the umbilicus, 16 cm superior to theumbilicus to 16 cm inferior to the umbilicus, 15 cm superior to theumbilicus to 15 cm inferior to the umbilicus, 14 cm superior to theumbilicus to 14 cm inferior to the umbilicus, 13 cm superior to theumbilicus to 13 cm inferior to the umbilicus, 12 cm superior to theumbilicus to 12 cm inferior to the umbilicus, 11 cm superior to theumbilicus to 11 cm inferior to the umbilicus, 10 cm superior to theumbilicus to 10 cm inferior to the umbilicus, 9 cm superior to theumbilicus to 9 cm inferior to the umbilicus, 8 cm superior to theumbilicus to 8 cm inferior to the umbilicus, 7 cm superior to theumbilicus to 7 cm inferior to the umbilicus, 6 cm superior to theumbilicus to 6 cm inferior to the umbilicus, 5 cm superior to theumbilicus to 5 cm inferior to the umbilicus, 4 cm superior to theumbilicus to 4 cm inferior to the umbilicus, 3 cm superior to theumbilicus to 3 cm inferior to the umbilicus, 2 cm superior to theumbilicus to 2 cm inferior to the umbilicus, and 1 cm superior to theumbilicus to 1 cm inferior to the umbilicus and encircling the subject,and circular regions radiating up to 30 cm from the umbilicus.

A subject at risk for ischemia-related tissue damage may topicallyself-administer a TRP family receptor agonist to a predetermined regionor the TRP family receptor agonist may be administered by firstresponders or in an emergency room prior to clinical confirmation ofischemia. A subject at risk for ischemia-related tissue damage maytopically self-administer a TRPV1 agonist to a predetermined region orthe TRPV1 agonist may be administered by first responders or in anemergency room prior to clinical confirmation of ischemia.

Suitable time frames for topically administering a TRP family receptoragonist or TRPV1 agonist may include, but are not limited to, uponidentification that a subject is at risk for ischemia-related tissuedamage, after identification of an ischemia-related symptom, concomitantwith reestablishment of blood flow, or up to three hours afterreestablishment of blood flow. Additional suitable time frames mayinclude but are not limited to within 90, 80, 70, 60, 55, 50, 45, 40,35, 30, 25, 20, 15, 10, 5 or 1 minute after identification of anischemia-related symptom and up to 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 120, 150, or 180 minutes afterreestablishment of blood flow. It is recognized that TRP family receptorstimulation may inhibit damage from an ischemia related injury orcontinue to provide a cardioprotective effect after the stimulationoccurs. A late stage cardioprotective effect may occur up to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52 or more hours post TRP familyreceptor stimulation.

A subject at risk for angina may topically administer a TRP familyreceptor agonist to a predetermined region or the TRP family receptoragonist may be administered by a medical services provider. A subject atrisk for angina may topically self-administer a TRPV1 agonist to apredetermined region or the TRPV1 agonist may be administered by amedical services provider.

Suitable time frames for topically administering a TRP family receptoragonist to a subject at risk for angina may include, but are not limitedto chronically or intermittently administering the agonist. Bychronically is intended a pre-determined dosage schedule such as hourly,daily, weekly, monthly, bimonthly, quarterly, semiannually, annually,biennially, or continuously through a time release or dosage releasemethod, and administration related to an angina symptom inducing factor.It is recognized that continuous administration encompassesreplenishment, exchange, replacement, reintroduction, change, andremoval and replacement of the agonist delivery system.

Reestablishment of blood flow may occur without intervention or as aresult of medical intervention including but not limited to surgical andpharmacological interventions. Any means of reestablishing blood flowknown in the art may be used in the methods of the invention. Means ofreestablishing blood flow may include, but are not limited to, stentrelated interventions, coated stent insertions, angioplasty,percutaneous transluminal angioplasty, laser catheterization,atherectomy catheterization, TEC, DVI, angioscopy, radiationcatheterization, intravascular ultrasound, rotational atherectomy,radioactive balloons, heatable wires, heatable balloons, biodegradablestent struts, biodegradable sleeves, bypass surgery, angioplasty, bloodthinners, tissue plasminogen activator, thrombolytic medications,ultrasound, transcranial Doppler ultrasound, carotid endarterectomy,defibrillation, beta blockers, antiarrhythmic drugs, implantation of adefibrillator, valve replacement surgery, cardiac transplantation, ACEinhibitors, vasodilators, aspirin, clopidogrel, ticlopidine, andstreptokinase.

The TRP family may include, but is not limited to, the TRPV, TRPA, TRPM,TRPN, TRPP, TRPVm and TRPC subfamilies. The TRPV family or vanilloidsubfamily of heat-sensitive transient receptor potential (TRP) ionchannels, may include, but is not limited to TRPV1, TRPV2, TRPV3, andTRPV4. See for example, Gharat & Szallasi (2008) Expert Opin. Ther.Patents 18(2):159-209, herein incorporated by reference in its entirety.TRPV1 is also known as capsaicin vanilloid receptor-1 (VR1), capsaicinreceptor, type 1 vanilloid receptor, and vanilloid receptor.

A “vanilloid” is capsaicin or any capsaicin analogue that comprises aphenyl ring with two oxygen atoms bound to adjacent ring carbon atoms(one of which carbon atoms is located para to the point of attachment ofa third moiety that is bound to the phenyl ring). A vanilloid is a TRPV1ligand if it binds to TRPV1 with a K_(i) that is no greater than 10 μM.In various embodiments the K_(i) is no greater than 10 μM, no greaterthan 1 μM, no greater than 100 nM, or no greater than 10 nM in areceptor binding assay.

The TRPM and TRPA subfamilies include receptors known as cold receptors.TRPM8 and TRPA1 are cold receptors that may function in protection fromischemia related tissue damage. Cold activation of TRPM8 or TRPA1 mayoccur through application of a chemical cold patch such as but notlimited to patches comprising menthol. Rapid cold producing substancesare known in the art.

An agonist means any compound including, but not limited to, smallmolecules, polypeptides, peptides, agents, and antibodies that activatesa receptor.

As used herein a TRP family receptor agonist refers to a compound oragent that elevates an activity of a TRP family receptor above the basalactivity level of the receptor. A TRP family receptor agonist activatesat least one member of the TRP family of receptors. A TRPV familyreceptor agonist refers to a compound or agent that elevates an activityof a TRPV family receptor above the basal activity level of thereceptor. A TRPV family receptor agonist refers to a compound or agentthat elevates an activity of a TRPV family receptor above the basalactivity level of the receptor. As used herein a TRPV1 agonist refers toa compound or agent that elevates an activity of TRPV1 above the basalactivity level of the receptor. TRPV1 agonists useful in the methods mayinclude a particular compound or agent that may function to elevate anactivity of TRPV1 while not affecting another TRPV1 activity. TRPV1agonists useful in the methods may include selective agonists. A“selective agonist” means that the agonist generally has greater,activity toward a certain receptor(s) compared with other receptors; theselective agonist need not be completely inactive with regards to theother receptors. Methods of assessing activation of TRPV1 may includewhole cell patch clamp analysis, ratiometric calcium imaging, excisedpatch clamp analysis, whole cell tail current analysis, calciummobilization assay, cultured dorsal root ganglion assay, calcium influxassays, and in vivo pain relief assay. TRPV1 binding assays andcompetition binding assays may provide additional information about aTRPV1 agonist. See for example 2006/0194805, herein incorporated byreference in its entirety.

TRPV1 activities may include, but are not limited to, triggering C fibermembrane depolarization, opening a cation selective channel, opening aCa²⁺ ion channel, opening a Na⁺ ion channel, opening a Ca²⁺/Na⁺ ionchannel, opening a non-selective ion channel. See for example2006/0194805, herein incorporated by reference in its entirety.

TRPV1 agonists may include, but are not limited to: capsaicin, piperine,rutaecarpine, capsaicinoids, resiniferatoxin (RTX),N-vanillylnonanamides, N-vanillylsulfonamides, N-vanillylureas,N-vanillylcarbamates, N[(substituted phenyl)methyl]alkylamides,methylene substituted [(substituted phenyl)methyl]alkenamides,N[(substituted phenyl)methyl]-cis-monosaturated alkenamides,N[(substituted phenyl)methyl]diunsaturated amides, 3-hydroxyacetanilide;hydroxyphenylacetamides, pseudocapsaicin, dihydrocapsaicin,nordihydrocapsaicin anandamide, zingerone, warburganal, polygodial,aframodial, cinnamodial, cinnamosmolide, cinnamolide, isovelleral,scalaradial, ancistrodial, β-acaridial, α-acaridial, merulidial,scutigeral, zucapsaicin, nonivamide, nicoboxil, davasaicin,resiniferotoxin, 3-hydroxyacetanilide, anandamide, olvanil, cannabidiol,civamide, eugenol, guaiacol, vanillotoxins, VaTx1, VaTx2, VaTx3,DA-5018, tinyatoxins, spider toxins, arvanil, O-1861,N-arachidonoyldopamine (NADA), phenylacetylrinvanil, O-2142,N-oleoyl-dopamine, and oleo-ethanolamide, and capsaicin receptor agonistcompounds of Szallasi & Blumberg (1999) Pharma Reviews 51:159-211,WO04007495, Gharat & Szallasi (2008) Expert Opin Ther. Patents18(2):159-209, Starowicz et al (2008) Current Pharmaceutical Design14:42-54; and Cromer & McIntyre (2008) Toxicon 51:163-173, hereinincorporated by reference in their entirety. Additional exemplary TRVP1agonists are described in U.S. Pat. Nos. 4,599,342; 5,962,532;5,221,692; 4,313,958; 4,532,139; 4,544,668; 4,564,633; 4,544,669;4,493,848; 4,544,668; and those described in 20050090557, 20030104085,and 20060194805. Mixtures of agonists and pharmaceutically acceptablesalts of any of the foregoing may also be used.

A “pharmaceutically acceptable salt” of a compound recited herein is anacid or base salt that is suitable for use in contact with the tissuesof human beings or animals. Such salts include mineral or organic acidsalts of basic residues such as amines, as well as alkali or organicsalts of acidic residues such as carboxylic acids. Pharmaceuticallyacceptable salts of compounds are known in the art. See for example,Berge et al (2006) J. Pharmaceutical Sciences 66:1-19; Stahl & WermuthEd (2008) Pharmaceutical Salts: Properties, Selection and Use Verlag;herein incorporated by reference in their entirety.

Capsaicinoids or capsaicin analogues may exhibit similar physiologicalproperties to capsaicin such as for example, trigger C fiber membranedepolarization by opening of cation channels permeable to calcium andsodium. Potency of a capsaicinoid may differ significantly fromcapsaicin. Capsaicinoids may include, but are not limited to,resiniferatoxin, and compounds described in U.S. Pat. Nos. 5,290,816;4,812,446; and 4,424,205; WO 96/40079; EPO 149 545; and in Ton et al(1955) Brit. J. Pharm 10:175-182; herein incorporated by reference intheir entirety. Other capsaicinoids may include, but are not limited to,N-vanillylnonanamides, N-vanillylsulfonamides, N-vanillylureas,N-vanillylcarbamates, N[(substituted phenyl)methyl]alkylamides,methylene substituted [(substituted phenyl)methyl]alkanamides,N[(substituted phenyl)methyl]-cis-monosaturated alkenamides,N[(substituted phenyl)methyl]diunsaturated amides, 3-hydroxyacetanilide,hydroxyphenylacetamides, pseudocapsaicin, dihydrocapsaicin,nordihydrocapsaicin, homocapsaicin, homodihydrocapsaicin I, anandamide,piperine, zingerone, warburganal, polygodial, aframodial, cinnamodial,cinnamosmolide, cinnamolide, civamide, nonivamide, olvanil,N-oleyl-homovanillamidia, isovelleral, scalaradial, ancistrodial,β-acaridial, merulidial, and scutigeral.

TRPV2 agonists may include, but are not limited to, probenecid.

TRPV3 agonists may include, but are not limited to incensole acetate.

TRPV4 agonists may include, but are not limited to, 4 α-phorbol12,13-didecanoate

“Therapeutically effective amount” or “therapeutically effective dose”refers to the quantity or dose of an agent required to produce aclinically desired result such as a biological or chemical response(such as, but not limited to, inhibition of ischemia-related tissuedamage in a subject in need of such inhibition), alleviation oramelioration of one or more symptoms of a disease or condition,diminishment of extent of disease, or prevention of damage.

A therapeutically effective amount of capsaicin may be within the rangeof 10 μg to 10 g capsaicin/kg subject, 10 μg to 5 g capsaicin/kgsubject, 10 μg to 3 g capsaicin/kg subject, 10 μg to 2 g capsaicin/kgsubject, 10 μg to 1 g capsaicin/kg subject, 10 μg to 500 mg capsaicin/kgsubject, 10 μg to 400 mg capsaicin/kg subject, 10 μg to 300 mgcapsaicin/kg subject, 10 μg to 200 mg capsaicin/kg (subject), 20 μg to200 mg capsaicin/kg (subject), 30 μg to 190 mg capsaicin/kg (subject),40 μg to 180 mg capsaicin/kg subject, 50 μg to 180 mg capsaicin/kgsubject, 60 μg to 180 mg capsaicin/kg subject, 70 μg to 180 mgcapsaicin/kg subject, 80 μg to 180 mg capsaicin/kg subject, 90 μg to 180mg capsaicin/kg subject, 0.1 mg to 180 mg capsaicin/kg subject, 0.1 mgto 170 mg capsaicin/kg subject, 0.1 mg to 160 mg capsaicin/kg subject,0.1 mg to 150 mg capsaicin/kg subject, 0.1 mg to 140 mg capsaicin/kgsubject, 0.1 mg to 130 mg capsaicin/kg subject, 0.1 mg to 120 mgcapsaicin/kg subject, 0.1 mg to 110 mg capsaicin/kg subject, 0.1 mg to100 mg capsaicin/kg subject, 0.1 mg to 90 mg capsaicin/kg subject, 0.1mg to 80 mg capsaicin/kg subject, 0.1 mg to 70 mg capsaicin/kg subject,0.1 mg to 60 mg capsaicin/kg subject, 0.1 mg to 50 mg capsaicin/kgsubject, 0.1 mg to 40 mg capsaicin/kg subject, 0.1 mg to 30 mgcapsaicin/kg subject, 0.1 mg to 30 mg capsaicin/kg subject, 0.2 mg to 30mg capsaicin/kg subject, 0.3 mg to 30 mg capsaicin/kg subject, 0.4 mg to30 mg capsaicin/kg subject, 0.5 mg to 30 mg capsaicin/kg subject, 0.6 mgto 30 mg capsaicin/kg subject, 0.7 mg to 30 mg capsaicin/kg subject 0.8mg to 30 mg capsaicin/kg subject, 0.9 mg to 30 mg capsaicin/kg subject,1 mg to 30 mg capsaicin/kg subject, preferably 1 mg to 20 mgcapsaicin/kg (subject), and 9 mg-11 mg capsaicin/kg subject. Theconcentration of capsaicin in a gel may be within the range of0.01%-15%, 0.05%-12.5%, 0.05%-10%, 0.1%-10%, 0.1%-9%, 0.1%-8%, 0.1%-7%,0.1%-6%, 0.1%-5%, 0.1%-4%, 0.1%-3%, 0.1%-2%, or 0.1%-1%.

The capsaicin compounds utilized in the methods and kits provided hereinmay include, but are not limited to USP-grade capsaicin,trans-capsaicin,(6E)-N-[[(4-Hydroxy-3-methoxyphenyl)methyl]-8-methyl-6-nonenamide andrelated capsaicinoids.

“Topically administering”, “topical”, and grammatical equivalentsthereof, refer to administering a biologically active compound orelectrical stimulation to a pre-defined, pre-determined, definite area,defined, or limited area of the skin. “Topical”, “transcutaneous”,“transdermal”, “transdermic” and “percutaneous” are intended to indicateunbroken skin. Thus, topically administering as used herein does notinclude administering by subcutaneous injection.

For topical administration, the TRP family receptor agonist including,but not limited to a TRPV agonist, may be formulated by any means knownin the art for direct application to a target area such as but notlimited to a region extending from thirty centimeters superior to theumbilicus to thirty centimeters inferior to the umbilicus and encirclingthe subject. Forms chiefly conditioned for topical application mayinclude, but are not limited to, creams, milks, gels, dispersion ormicroemulsions, lotions, impregnated pads, ointments, or sticks, andaerosol formulations such as, but not limited to, sprays or foams,pastes, jellies and patches. The compositions may be formulated asaqueous compositions or may be formulated as emulsions of one or moreoil phases in an aqueous continuous phase. Thus the TRP family receptoragonist may be delivered via patches, bandages, cloths, tissues, swabs,sticks, or brushes. Alternatively the agent may be formulated to be partof an adhesive polymer such as polyacrylate or acrylate/vinyl acetatecopolymer.

As described above a TRP family receptor agonist including, but notlimited to a TRPV agonist, may be delivered via patches for transdermaladministration. See U.S. Pat. No. 5,560,922 for examples of patchessuitable for transdermal delivery of a therapeutic agent. Patches fortransdermal delivery may comprise a backing layer and a polymer matrixwhich has dispersed or dissolved therein a therapeutic agent, and may ormay not contain one or more skin permeation enhancers. The backing layercan be made of any suitable material which is impermeable to thetherapeutic agent. The backing layer serves as a protective cover forthe matrix layer and provides also a support function. The backing canbe formed so that is essentially the same size layer as the polymermatrix or it can be of larger dimension so that it can extend beyond theside of the polymer matrix and then may extend outwardly in a mannerthat the surface of the extension of the backing layer may be the basefor an adhesive means. Alternatively, the polymer matrix may contain orbe formulated with an adhesive polymer such as polyacrylate oracrylate/vinyl acetate copolymer.

Examples of materials suitable for making the backing layer may include,but are not limited to, films of high and low density polyethylene,polypropylene, polyurethane, polyvinylchloride, polyesters such aspoly(ethylene phthalate), metal foils, metal foil laminates of suchsuitable polymer films and the like. Preferably the materials used forthe backing layer are laminates of such polymer films with a metal foilsuch as aluminum foil. In such laminates, a polymer film of the laminatewill usually be in contact with the adhesive polymer matrix.

The backing layer can be any appropriate thickness which will providethe desired protective and support functions. A suitable thickness maybe from about 10 to about 200 microns. In an embodiment, a patch fortransdermal delivery of a TRP family receptor agonist may be providedwith a placement guide. A placement guide facilitates administration atthe appropriate pre-determined site. A placement guide may bestructural, pictorial, verbal or a combination thereof. The placementguide may be integral to the patch, removable from the patch or separatefrom the patch.

Generally those polymers used to form the biologically acceptableadhesive polymer layer are those capable of forming shaped bodies, thinwalls or coatings through which therapeutic agents can pass at acontrolled rate. Suitable polymers are biologically and pharmaceuticallycompatible, nonallergenic and insoluble in and compatible with bodyfluids or tissues with which the device is contacted.

Exemplary materials for fabricating the adhesive polymer layer includepolyethylene, polypropylene, polyurethane, ethylene/propylenecopolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetatecopolymers, silicone elastomers, especially the medical-gradepolydimethylsiloxanes, neoprene rubber, polyisobutylene, polyacrylates,chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinylacetate copolymer, cross-linked polymers (hydrogel), polyvinylidenechloride, poly(ethylene terephthalate), butyl rubber, epichlorohydrinrubbers, ethylenvinyl alcohol copolymers, for example,polysiloxane-polycarbonate copolymers, polysiloxane-poly-ethylene oxidecopolymers, polysiloxane-polymethacrylate copolymers,polysiloxane-alkylene copolymers (e.g. polysiloxane-ethylenecopolymers), polysiloxane-alkylenesilane copolymers (e.g.polysiloxane-ethylenesilane copolymers), and the like; cellulosepolymers for example methyl or ethyl cellulose; hydroxylpropyl methylcellulose, and cellulose esters; polycarbonates,polytetrafluoroethylene, and the like.

Preferably, a biologically acceptable adhesive polymer matrix should beselected from polymers with glass transition temperatures below roomtemperature. The polymer may, but need not necessarily, have a degree ofcrystallinity at room temperature. Cross-linking monomeric units orsites can be incorporated into such polymers. For example, cross-linkingmonomers can be incorporated into polyacrylate polymers, which providesites for cross-linking the matrix after dispersing the therapeuticagent into the polymer. Known cross-linking monomers for polyacrylatepolymers include polymethacrylic esters of polyols such as butylenediacrylate and dimmethacrylate, trimethylol propane trimethacrylate andthe like. Other monomers which provide such sites include allylacrylate, allyl methacrylate, diallyl maleate and the like.

A plasticizer and/or humectant may be dispersed within the adhesivepolymer matrix. Water-soluble polyols may be suitable for this purpose.Incorporation of a humectant in the formulation allows the dosage unitto absorb moisture on the surface of the skin which in turn helps toreduce skin irritation and to prevent the adhesive polymer layer of thedelivery system from failing.

Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and maycontain one or more emulsifying agents, stabilizing agents, dispersingagents, suspending agents, thickening agents, or coloring agents. Thepercent by weight of a therapeutic agent of the invention present in atopical formulation may depend on various factors, but generally may befrom about 0.01% to 95% of the total weight of the formulation, andtypically 0.1 to 85% of the total weight of the formulation.

In various embodiments the TRP family receptor agonist including, butnot limited to a TRPV agonist, may be topically administered using asponge, aerosol, spray, brush, swab, or other applicator. The applicatormay provide either a fixed or variable metered dose application such asa metered dose aerosol, a stored-energy metered dose pump, or manualmetered dose pump. The applicator may have measuring marks for assistinga user in determining the amount of the composition in the applicatordevice.

Modulating may be altering, changing, varying, affecting, adjusting, orregulating something of interest, such as ischemia-related tissuedamage, tissue damage, or a cardiac tissue damage characteristic.Modulating may encompass improving a cardiac tissue damagecharacteristic or increasing or decreasing a cardiac tissue damagecharacteristic by at least 1%, 5%, preferably 10%, 20%, more preferably30%, 40%, 50%, 60%, yet more preferably 70%, 80%, 90%, or 100% ascompared to an untreated or placebo treatment effect. “Improving acardiac tissue damage characteristic” means changing, altering, moving,shifting or returning a tissue damage characteristic to a state or levelcloser to that found in a subject not undergoing an ischemia-relatedevent such as a healthy or normal subject. Any method of evaluating atissue damage characteristic known in the art may be used in theprovided methods. It is recognized that the effects of the methodsprovided herein may be compared to an untreated or placebo effect in anindividual or population prior to the use of the methods or kits.

Cardiac tissue damage characteristics may include, but are not limitedto, altered cardiac function, altered ECG patterns, arrhythmias,dysrrhythmias, left ventricular (LV) function, LV systolic functionparameters such as but not limited to, left ventricular dimension,stroke volume index, LV fractional shortening, LV ejection fraction, orpeak systolic strain; diastolic and systolic dimensions,intraventricular septum thickness, LV posterior wall thickness, leftatrial dimension, ejection fraction and altered cardiac biomarkers.

Methods of evaluating cardiac function or cardiac viability may include,but are not limited to, serial echocardiogram, two dimensional speckletracking imaging, transthoracic echocardiogram, echo-Doppler,electrocardiogram (ECG), serial ECG, continuous ECG, stress testing,electrophysiological testing, tilt table testing, radiologicalprocedures, x-rays, ultrasonography, M-mode ultrasound, two-dimensionalultrasound, Doppler ultrasound, color Doppler, echocardiography, MRI,magnetic resonance angiography (MRA), radionuclide imaging, positronemission tomography (PET), cardiac catheterization, central venouscatheterization, angiography, cineangiography, computed tomography (CT),computed tomography angiography (CTA), fluoroscopy, hematoxylin & eosinstaining, annexin staining, and TUNEL staining.

Cardiac biomarkers may include, but are not limited to troponin, serumcreatine kinase, plasma CGRP, blood urea nitrogen, serum creatinine,serum potassium, serum sodium, serum chloride, serum bicarbonate,troponin T, troponin I, CK-MB, myoglobin, and NT-proBNP. Any method ofevaluating a cardiac biomarker known in the art may be used in themethods of the invention.

Any method of performing an imaging modality known in the art may beutilized in the methods and kits provided herein. Imaging modalities mayinclude, but are not limited to, serial echocardiogram, two dimensionalspeckle tracking imaging, transthoracic echocardiogram, echo-Doppler,electrocardiogram (ECG), serial ECG, continuous ECG, x-rays,ultrasonography, M-mode ultrasound, two-dimensional ultrasound, Dopplerultrasound, color Doppler, echocardiography, MRI, magnetic resonanceangiography (MRA), radionuclide imaging, positron emission tomography(PET), cardiac catheterization, central venous catheterization,angiography, cineangiography, computed tomography (CT), computedtomography angiography (CTA), and fluoroscopy.

“Biological sample” means a sample collected from a subject including,but not limited to, whole blood, serum, tissue, cells, mucosa, fluid,scrapings, hairs, cell lysates, urine, and secretions. Biologicalsamples such as serum samples can be obtained by any method known to oneskilled in the art. Further, biological samples can be enriched,purified, isolated, or stabilized by any method known to one skilled inthe art. Such enrichment, purification, isolation, or stabilizationprocedures can be performed at any time during the methods of theinvention. A person skilled in the art of obtaining biological sampleswould recognize various benefits and drawbacks associated with thedifferent methodologies and would choose accordingly.

Kits provided herein may comprise a TRP family receptor agonist such asbut not limited to, a TRPV agonist, in one or more dosage unit forms foruse in the methods and usage instructions. Preferredly the usageinstructions will indicate the appropriate predetermined location fortopical administration of the TRPV1 agonist for the type of tissuedamage that the subject is at risk for. It is envisioned that the dosageunit form may be optimized for the predetermined location appropriatefor a particular condition or indication.

“Dosage unit form” as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

Organ transplantation may include, but is not limited to, removal of anorgan, portion of an organ, or tissue from a donor subject andre-implantation of the organ or portion of an organ in a recipientsubject. A donor subject is a subject from which the organ, portion ofan organ, or tissue is removed. The donor subject may be a livingsubject (as for example with a kidney transplant) or a dead subject. Arecipient subject is a subject in which the organ, portion of an organ,or tissue is implanted, grafted, or placed. In an embodiment, the donorsubject and the recipient subject may be the same individual, as forinstance, with a muscle or skin graft. The methods encompassadministering a TRP family receptor agonist such as, but not limited toa TRPV1 agonist, to at least one of the donor and recipient subjectsprior to or concomitant with the organ transplantation procedure. Theorgan transplantation procedure has multiple components and it isrecognized that the time frames suitable for administering a a TRPfamily receptor agonist such as, but not limited to a TRPV1 agonist to adonor subject may differ from those suitable for a recipient subject.Components of the organ transplantation procedure may include, but arenot limited to, preparation of the donor subject, removal of the desiredorgan, organ portion, or tissue from the donor, transportation of thedonated organ, organ portion, or tissue to the recipient, preparation ofthe recipient subject, and implantation, grafting, or connecting of thedonated organ, organ portion or tissue.

Methods and kits provided herein may include methods and kits forischemia-related tissue damage in a subject at risk for ischemia-relatedtissue damage comprising the steps of identifying a subject at risk forischemia-related tissue damage and topically administering atherapeutically effective amount of a C-fiber stimulator. C-fiberstimulators may include, but are not limited to, TRP family receptoragonists, TRPV family receptor agonists, TRPV1 agonists, capsaicin,capsaicin analogues and electrical stimulation. C-fibers, C sensoryfibers, and C fiber nociceptors may be used interchangeably herein andrefer to a type of peripheral pain carrying nerve fiber. C-fibersinclude both TRPV1 positive C-fibers and TRPV1 negative C-fibers. TRPfamily pain receptor channels including, but not limited to the TRPV1pain receptor channel, are activated by transdermal electricalstimulation. Transdermal electrical stimulation may activate one or moreTRP family pain receptor channels. Electrical stimulation that may beutilized in the provided methods may include, but is not limited to,transcutaneous electrical stimulation and electroacupuncture.Therapeutically effective electrical stimulation may include up to 1, 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes of pulsingtranscutaneous electrical stimulation. Such pulsing may be within therange of 1-30 volts, 2-20 volts, 3-19 volts, 4-18 volts, 5-17 volts,6-16 volts, 7-15 volts, 8-14 volts, 8-12 volts, and 9-11 volts. Theduration of the pulse may range from 0.1-2 ms, 0.1-1.5 ms, 0.1-1 ms,0.1-0.9 ms, 0.1-0.8 ms, 0.1-0.7 ms, 0.2-0.6 ms, 0.3-0.6 ms, and 0.4-0.6ms. The frequency may range from 1-500 Hz, 10-400 Hz, 20-300 Hz, 30-200Hz, 40-170 Hz, 50-140 Hz, 60-130 Hz, 70-120 Hz, 80-110 Hz, and 90-105Hz.

The following examples are offered by way of illustration and notlimitation.

EXPERIMENTAL Example 1 Mice

Wild-type (B6129SF2/J F2) and bradykinin 2 receptor knockout (BK2R KO)mice (B6129 SF2/J F2, strain 101045) were obtained from JacksonLaboratories (Bar Harbor, Me.). Mice were maintained in accordance withinstitutional guidelines, the Guide for the Care and Use of LaboratoryAnimals (NIH, revised 1985) and the Position of the American HeartAssociation on Research Animal Use (1984). All groups of mice consistedof males and females distributed equally among groups; post-hoc analysisconfirmed previous results that there were no gender related differencesin these studies.

Example 2 Mouse Model of Ischemia and Reperfusion

A previously described mouse model of ischemia and reperfusion (I/R)injury in vivo was used (Ren et al. (2004) J. Surg Res. 121(1):120-129;Fan et al (2005) Circulation 111(14):1792-1799; Zhou et al (2007)Cardiovascular Research 75(3) 487-497; and Diwan et al (2007) J.Clinical Investigation 117(10):2825-2833, herein incorporated byreference in their entirety). Mice were anesthetized with sodiumpentobarbital (90 mg/kg ip), intubated with PE 90 tubing and ventilatedusing a mouse miniventilator (Harvard Apparatus, Holliston, Mass.). Allmice were continuously monitored by electrocardiography (ECG) (DigiMedSinus Rhythm Analyzer, Micro-Med Inc), and mice without evidence ofischemia and timely reperfusion were excluded from the studies.

A lateral thoracotomy (1.5 cm incision between the second and third ribswas performed to provide exposure of the left anterior descendingcoronary artery (LAD). Vascular bundles in the vicinity were coagulatedusing a microcoagulator (Medical Industries Inc, St. Petersburg Fla.).An 8-0 nylon suture was placed around the LAD 2-3 mm from the tip of theleft auricle and a piece of soft silicon tubing (0.64 mm ID, 1.19 mm OD)was placed over the artery. Coronary occlusion was achieved bytightening and tying the suture. Coronary occlusion was for 45 minutes,unless indicated otherwise. At the end of the occlusion, the suture wasuntied and left in place. Ischemia was confirmed by visual observationof cyanosis and by continuous ECG monitoring (QRS complex widening, Twave inversion and ST segment changes); reperfusion was confirmed byreversal of these effects. Results of one such experiment are presentedin FIG. 1, panel A.

Example 3 Infarct Size Determination and Imaging

Mice were euthanized either 4 or 24 hours after reperfusion and infarctsize determination and imaging were performed by previously describedmethods (Ren et al. (2004) J. Surg Res. 121(1):120-129 and Guo et al(1998) Am J. Physiology 275 (4 Pt 2):H1375-1387, herein incorporated byreference in their entirety). Mice were anesthetized with pentobarbitalsodium (35 mg/kg iv) and euthanized with an intravenous bolus of KCl.The heart was excised and perfused with Krebs-Henseleit solution throughan aortic cannula (22 or 23-gauge needle) using a Langerdorff apparatus.To delineate infarcted from viable myocardium, the heart was thenperfused with a 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC)in phosphate buffer (pH 7.4, 37° C.) at a pressure of 60 mM Hg (approx.3 ml over 3 min). To delineate the occluded-reperfused coronary vascularbed, the coronary artery was then tied at the site of the previousocclusion and the aortic root was perfused with a 5% solution of phthaloblue dye in normal saline. The portion of the left ventricle (LV)supplied by the previously occluded coronary artery (risk region) wasidentified by the absence of blue dye, whereas the rest of the LV wasstained dark blue. The heart was frozen. Atrial and right ventriculartissues were excised. The LV was sectioned into 5-7 transverse sliceswhich were fixed in 10% neutral buffered formaldehyde. The sections werephotographed using a Nikon Coolpix880 digital camera fitted with UR-E2macro lens and computerized digital planimetry was performed using NIHImage software. Infarct size was determined by the method of Fishbein etal (1981) Am Heart J 101(5):593-600, herein incorporated by reference inits entirety. The four hour time point was used only in the spinaltransection and related control studies.

Example 4 Abdominal and Skin Incisions

Two cm abdominal incisions were made through the skin, subcutaneous,fat, muscle, and peritoneum at the abdominal midline. For skinincisions, the incision was made anatomically in the same location butcare was taken to cut the skin only. Control mice were intubated,shaved, and the skin was sterilized but no incision was made.

Example 5 Spinal Cord Transection

Spinal cord transection was used to investigate the involvement of thecentral nervous system in the cardioprotective effect of TRPV1activation. Mice were anesthetized and bone markers were identified atC7 and T7 vertebra levels. The spinal cords of cervical 7 (C7) andthoracic 7 (T7) were transected by a sharp knife with limited bleedingunder a microscope followed by immediate abdominal incision. Fifteenminutes later, the mice were subjected to the surgicalischemia/reperfusion protocol described above herein. Fours hours afterreperfusion, mice were euthanized and infarct size determination wasperformed without the mice regaining consciousness. Results from onesuch experiment are presented in FIG. 2, panel B.

Example 6 Neuronal Tracing

A fluorescent dye1,1′-dioactadecyl-3,3,3′,3′-tetramethylindocarbodyanine perchlorate(Dil) was injected subcutaneously at the abdominal incision level(thoracic vertebra T9-T10) of the spine. One week after injection, micewere perfused with 4% formaldehyde. The fixed spinal cord and dorsalroot ganglia at thoracic vertebra T9-T10 and T1-T5 levels weredissected. The sectioned spinal cord (40 μm) and whole mount dorsal rootganglion were visualized by confocal microscopy. Images captured fromone such experiment are shown in FIG. 2, panel C.

Example 7 Morphological and Histological Assessment of Cardiac Tissue

Hematoxylin and eosin staining were performed to evaluate cardiac tissueafter exposure to surgically induced ischemia and reperfusion. Mice wereeuthanized 24 hours after surgery. Hearts were perfused with PBS thenfixed by perfusion with 10% formalin in PBS. Hearts were post-fixedovernight, embedded in paraffin, sectioned, stained, and examinedmicroscopically. Results from one such experiment are presented in FIG.1, E.

Example 8 Evaluation of Apoptosis

Mice were euthanized 24 hours after surgery. Hearts were perfused withPBS then fixed by perfusion with 10% formalin in PBS. Hearts werepost-fixed overnight, embedded in paraffin and sectioned. In situ DNAfragmentation was assessed using the DeadEnd™ Fluorometric TUNEL system(Promega), followed by staining with anti-sarcomeric actin antibody(Sigma) and DAPI (Invitrogen). TUNEL-positive (green) nuclei werecounted from 10 randomly chosen microscopic fields of the midventricularsection (n=5) and were expressed as a percentage of total nuclei (bothblue and green staining nuclei; approximately 400 nuclei counted perfield) in the same fields. Results from one such experiment arepresented in FIG. 1.

Example 9 Evaluation of Apoptosis (2)

DNA fragmentation in cardiac tissue post-ischemia/reperfusion wasassessed by a cell-death detection ELISA kit (Roche Applied Science).The cell-death detection ELISA measures the content of cytosolic mono-and oligo-nucleosomes (180 base pair nucleotides or multiples) byemploying the sandwich enzyme immunoassay technique. Results werenormalized to the standard provided in the kit and expressed as afold-increase over the control.

Example 10 Remote Capsaicin Inhibition of Ischemia-Related Tissue Damage(1)

Gender matched mice were obtained. Experimental and sham (control)treated mice were shaved and prepped similarly. Capsaicin gel (0.1%capsaicin in a gel carrier, 200-250 μl) was applied to a 25 g mouse.This is approximately 10 mg/kg body weight. Fifteen minutes laterischemia and reperfusion were surgically induced as described elsewhereherein. The coronary occlusion was maintained for 45 minutes and thenreleased, allowing reperfusion and reestablishing blood flow. Infarctsize was determined as described elsewhere herein.

Example 11 Remote Capsaicin Inhibition of Ischemia-Related Tissue Damage(2)

Gender matched mice were obtained. Experimental and control treated micewere shaved and prepped similarly (n=6 mice per group). Capsaicin (0.1%capzacin cream) was administered over a 2 cm² skin area at the anteriorwall of the abdomen to mice in the experimental group. Fifteen minutesafter either sham treatment or administration of capsaicin, surgicalinduction of ischemia and reperfusion was performed as describedelsewhere herein. The coronary occlusion was maintained for 45 minutesand then released, allowing reperfusion and reestablishing blood flow.Infarct size was determined as described elsewhere herein. In at leastone experiment, the area of capsaicin application corresponded to theT10 level in humans.

Example 12 Remote Electrical Inhibition of Ischemia-Related TissueDamage

Age and gender matched mice were obtained. Experimental and controltreated mice were anesthetized, shaved, and electrodes were placedsimilarly (n=6 mice per group). Fifteen minutes of transcutaneouselectrical stimulation (TES) (10 volts, 0.4-0.5 ms pulse width, 100 Hz)was administered to mice in the experimental group. Fifteen minutesafter either sham treatment (wire placement but no current) orconclusion of TES, ischemia was induced by coronary occlusion asdescribed elsewhere herein. The coronary occlusion was maintained for 45minutes and then released, allowing reperfusion and reestablishing bloodflow. Infarct size was determined as described elsewhere herein.

Example 13 Remote Electrical Stimulation During Ischemia

Age and gender matched mice were obtained. Experimental and controltreated mice were anesthetized, shaved, and electrodes were placedsimilarly (n=6 mice per group). Ischemia was induced by coronaryocclusion as described elsewhere herein. The coronary occlusion wasmaintained for 30 minutes. Fifteen minutes of transcutaneous electricalstimulation (TES) (10 volts, 0.5 ms, 100 Hz, 400 pulses) wasadministered to mice in the experimental group. Fifteen minutes afterthe TES ended, the coronary occlusion was released, allowing reperfusionand reestablishing blood flow. Infarct size was determined as describedelsewhere herein. In at least one experiment, the area of electricalstimulation corresponded to the T10 level in humans

Example 14 Analysis of Role of C-Fibers in Inhibition ofIschemia-Related Tissue Damage

A relatively selective antagonist of peripheral C-fiber nociceptors,lidocaine, was tested. Ten μl lidocaine (2%) was administeredsubcutaneously (s.c.) to mice. Subsequently the mice received either asham treatment or remote electrical stimulation as described elsewhereherein. After 15 minutes, ischemia was induced by coronary occlusion.The occlusion was maintained for 45 minutes prior to reestablishment ofblood flow. Pre-treatment with lidocaine reduces the cardioprotectiveeffect of the remote electrical stimulation, such that there is nosignificant cardioprotection relative to controls. Results from one suchexperiment are presented in FIG. 2, panel A.

Example 15 Cell Fractionation and Immunoblotting

Mice were euthanized thirty minutes after either sham treatment orabdominal electrical stimulation. Hearts were excised and LV samples(0.5 g) were flash-frozen in liquid nitrogen until used. Frozen tissueswere powdered at liquid nitrogen temperature and homogenized in buffer A(5 mM Tris, 4 mM EGTA, 2 mM EDTA, 5 mM dithiothreitol, 1 mMphenylmethylsulfonyl fluoride and EDTA-free protease inhibitor (1tablet/10 ml)). The homogenate was centrifuged at 100,000×g for 30minutes at 4° C. The new supernatant was used as the cytosolic fraction:The pellet was re-homogenized with homogenization buffer A containing 1%Triton X, incubated on ice for twenty minutes and then centrifuged at100,000×g for 30 minutes at 4° C. The new supernatant was used as themembrane-associated fraction. Protein concentration was determined via aBio-Rad protein assay (Bio-Rad).

Cytosolic and membrane-associated fractions (50 μg) were electrophoresedon a 10% SDS-PAGE gel. After transfer to membranes, immuno-blottinganalysis was performed with PKC isoform specific primary antibodiesagainst PKCα, PKCδ, PKCε and PKCζ (Cell Signaling). The membrane wasnext incubated with HRP-conjugated secondary mouse (Bio-Rad) or rabbit(Santa Cruz) antibody. Membranes were developed using ECL-PLUS Westernblotting Detecting kit (Amersham Pharmacia Biotech) and densitometry ofprotein bands was quantified using a Fluorchem 880 gel imager. Transferefficiency and loading equality were examined by staining the membranewith 0.1% Ponceau S in 5% acetic acid. Stained membranes were digitallyscanned and densitometric analysis of major bands was used to normalizesignal intensity for quantitative analysis as described previously (Liaoet al (2007) J. Mol. Cell. Cardiology 42(1):106-120 and Brown et al(2005) Am. J. Physiol. 289(1):H466-476; herein incorporated by referencein their entirety). Results from one such experiment are presented inFIG. 7.

Example 16 Statistical Analysis

Group size was determined by Power analysis. For parameters thatrequired quantification and evaluation for statistical significance,results were expressed as mean±standard error of the mean (S.E.M.).Statistical significance (probability values) was determined using theStudent's t test (two tailed distribution and two sample unequalvariance) with the Bonferroni correction. For multiple groupcomparisons, one way analysis of variance (ANOVA) followed by Fisherpost hoc test was used. A P value≦0.05 was considered statisticallysignificant.

Example 17 Assessment of Topical TRPV Family Receptor Agonist Efficacyin Subject Exhibiting Stable Angina (I)

Subjects with chronic stable exertional angina or anginal equivalent whoare receiving background medical therapy are identified. Staffconducting and interpreting the exercise tests are blind to thetreatment applied to the subject. It is recognized that the burningsensation elicited by capsaicin will not allow the subjects to be blindfrom the subject's perspective.

Demographics, medical history, limited physical examinations, vitalsigns, ECG, and concomitant medication history are obtained fromprospective subjects. Prospective subjects undergo a small scalecapsaicin skin test. Capsaicin cream (0.15%) is applied to a 1×1 cm²area on the subject's forearm. The reaction and forearm area areobserved over the next 30 minutes. Subjects with severe reactions andsubjects without a burning sensation (indicating an intact TRPV1signaling axis) are excluded from the study.

Subjects are randomized. Half the subjects receive a capsaicin treatmentat the first visit; half receive a placebo at the first visit. At thesecond visit, the subject receives the alternate treatment. Subjects areasked to withhold anti-anginal medications the morning of each visit.

Subjects are treated with placebo or topical capsaicin cream (ChattemInc., 0.035%, 0.10%, or 0.15%) applied over an 8×15 cm² region centeredon the umbilicus (approximately 4 cm anterior and posterior and 7.5 cmleft and right of the umbilicus). The cream is applied with gloves andmassaged gently into the skin. The staff member applying the cream wearsan applicator mask. Thirty minutes (+/−3 minutes) after treatmentapplication, vitals and ECG are repeated. Forty-five minutes (+/−5minutes) after treatment application, subjects begin an exercise testprocedure. Subjects walk as long as they can tolerate, angina-relatedsymptom limited.

Onset of angina or angina-equivalent symptoms, descriptive quality, andintensity are recorded. Study medication is removed with alcohol swabs.Skin is assessed.

In 7 days (+/−1 day) subjects return for the second visit. The procedureis repeated but each subject receives the alternate treatment.

Exercise ECG date is interpreted by a cardiologist according to standardcriteria. Images are processed and analyzed using standard laboratorymethodology, including computer generated calculations of Summed StressScore (SSS) and Summed Difference Score (SDS).

Example 18 Assessment of Topical TRPV Family Receptor Agonist Efficacyin Subject Exhibiting Stable Angina (II): Myocardial Perfusion Imaging

Subjects are randomized as above. Subjects prepare for each visit asdescribed above. Resting imaging is performed on the subjects. The studymedicine is applied as described above herein.

An IV catheter is inserted in conjunction with the exercise test and aradioisotope is administered at rest and post exercise. Exercise testsare performed as described above herein. A Myoview, or Tc-99mTetrofosmin, scan is performed.

Example 19 Statistical Analysis of Angina Related Efficacy Results

Distributions of endpoints are examined graphically. Datatransformations are applied as necessary (e.g. logarithmictransformation of highly skewed data). If apparent violations ofparametric assumptions cannot be resolved, non-parametric procedures areapplied. All statistical tests and confidence intervals are based on amaximum type I error rate of α=0.05, two-sided.

Analysis of this two-period cross-over design is based on a three-factorrepeated measures analysis of variance (ANOVA) model. Treatment (twolevels) and study period (two levels) are within-subject factors, whilethe sequence of treatment administration (two levels) is a randomlyassigned between-subjects factor. If a significant carry-over (sequence)effect is observed, all possible estimates of the treatment differenceare computed (i.e. between patient-difference in the first period,between patient difference in the second period, within-patientdifference in the first sequence, within patient difference in thesecond sequence), but primacy is given to the between-patient differencein the first period.

Example 20 Duration of Cardioprotective Effect of C-Fiber StimulationAnalysis

Age and gender matched mice were obtained. Experimental and controltreated mice were anesthetized, shaved, and electrodes were placedsimilarly (n=6 mice per group). Fifteen minutes of transcutaneouselectrical stimulation (TES) (10 volts, 0.4-0.5 ms, 100 Hz, 400 pulses)was administered to mice in the experimental group. Twenty four hoursafter either sham treatment or conclusion of TES, ischemia was inducedby coronary occlusion as described elsewhere herein. The coronaryocclusion was maintained for 30 minutes and then released, allowingreperfusion and reestablishing blood flow. Infarct size was determinedas described elsewhere herein. Results from one such experiment arepresented in FIG. 12.

All publications, patents, and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents, and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually incorporated by reference.

Following from the above description, it should be apparent to those ofordinary skill in the art that, while the methods and kits hereindescribed constitute exemplary embodiments of the present invention, itis to be understood that the inventions contained herein are not limitedto the above precise embodiment and that changes may be made withoutdeparting from the scope of the invention. Likewise, it is to beunderstood that it is not necessary to meet any or all of the identifiedadvantages or objects of the invention disclosed herein in order to fallwithin the scope of the invention, since the invention is defined by theclaims and since inherent and/or unforeseen advantages of the claimedmethods and kits may exist even though they may not have been explicitlydiscussed herein.

That which is claimed:
 1. A method of inhibiting ischemia-relatedcardiac tissue damage in a human subject via remote sensory nervestimulation, the method comprising the steps of: (a) identifying a humansubject at risk for ischemia-related cardiac tissue damage; and (b)topically administering a therapeutically effective transcutaneouselectric stimulation to a predetermined region of skin selected from (1)a region of abdominal skin containing sensory nerves from which signaltravels to dorsal root ganglia at a thoracic vertebra level at or belowthe T7 level and encircling the subject and (2) partial regions withinsaid region of abdominal skin containing sensory nerves from whichsignal travels to dorsal root ganglia at a thoracic vertebra level at orbelow the T7 level and encircling the subject wherein saidtranscutaneous electric stimulation has a voltage of 1-30 volts and is apulsing electric stimulation adapted to remotely stimulate a sensorynerve, thereby inhibiting ischemia-related cardiac tissue damage.
 2. Themethod of claim 1, wherein administering said transcutaneous electricalstimulation occurs upon identifying said subject as being at risk forischemia-related cardiac tissue damage, after identification of anischemia-related symptom, concomitant with reestablishment of bloodflow, or up to three hours after reestablishment of blood flow.
 3. Themethod of claim 2, wherein administering said transcutaneous electricalstimulation occurs within fifteen minutes of the first identification ofan ischemia-related symptom.
 4. The method of claim 1, wherein thepredetermined region consists of a region of abdominal skin containingsensory nerves from which signal travels to dorsal root ganglia at theT9 to T10 thoracic vertebra level and encircling the subject.
 5. Themethod of claim 1, wherein the transcutaneous electric stimulation isapplied for a duration of at least about 15 minutes.